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In vivo delivery is a major barrier to the use of molecular tools for gene modification. Here we demonstrate site-specific gene editing of human cells in vivo in hematopoietic stem cell-engrafted NOD.Cg- Prkdc scid IL2rγ tm1Wjl (abbreviated NOD- scid IL2rγ null ) mice, using biodegradable nanoparticles loaded with triplex-forming peptide nucleic acids (PNAs) and single-stranded donor DNA molecules. In vitro screening showed greater efficacy of nanoparticles containing PNAs/DNAs together over PNA-alone or DNA-alone. Intravenous injection of particles containing PNAs/DNAs produced modification of the human CCR5 gene in hematolymphoid cells in the mice, with modification confirmed at the genomic DNA, mRNA and functional levels. Deep sequencing revealed in vivo modification of the CCR5 gene at frequencies of 0.43% in hematopoietic cells in the spleen and 0.05% in the bone marrow: off-target modification in the partially homologous CCR2 gene was two orders of magnitude lower. We also induced specific modification in the β-globin gene using nanoparticles carrying β-globin -targeted PNAs/DNAs, demonstrating this method’s versatility. In vivo testing in an enhanced green fluorescent protein- β-globin reporter mouse showed greater activity of nanoparticles containing PNAs/DNAs together over DNA only. Direct in vivo gene modification, such as we demonstrate here, would allow for gene therapy in systemic diseases or in cells that cannot be manipulated ex vivo .

There is a growing need for effective animal models to carry out experimental studies on human hematopoietic and immune systems without putting individuals at risk. Progress in development of small animal models for the in vivo investigation of human hematopoiesis and immunity has seen three major breakthroughs over the last three decades. First, CB17- Prkdc scid (abbreviated CB17- scid ) mice were discovered in 1983, and engraftment of these mice with human fetal tissues (SCID-Hu model) and peripheral blood mononuclear cells (Hu-PBL-SCID model) was reported in 1988. Second, NOD- scid mice were developed and their enhanced ability to engraft with human hematolymphoid tissues as compared with CB17- scid mice was reported in 1995. NOD- scid mice have been the “gold standard” for studies of human hematolymphoid engraftment in small animal models over the last 10 years. Third, immunodeficient mice bearing a targeted mutation in the IL-2 receptor common gamma chain ( IL2rγnull ) were developed independently by four groups between 2002 and 2005, and a major increase in the engraftment and function of human hematolymphoid cells as compared with NOD- scid mice has been reported. These new strains of immunodeficient IL2rg rγnull mice are now being used for studies in human hematopoiesis, innate and adaptive immunity, autoimmunity, infectious diseases, cancer biology, and regenerative medicine. In this chapter, we discuss the current state of development of these strains of mice, the remaining deficiencies, and how approaches used to increase the engraftment and function of human hematolymphoid cells in CB17- scid mice and in previous models based on NOD- scid mice may enhance human hematolymphoid engraftment and function in NOD- scid IL2r γnull mice.

IMP3 (insulin-like growth factor-2 mRNA binding protein 3) is an oncofetal protein whose expression is prognostic for poor outcome in several cancers. Although IMP3 is expressed preferentially in triple-negative breast cancer (TNBC), its function is poorly understood. We observed that IMP3 expression is significantly higher in tumor initiating than in non-tumor initiating breast cancer cells and we demonstrate that IMP3 contributes to self-renewal and tumor initiation, properties associated with cancer stem cells (CSCs). The mechanism by which IMP3 contributes to this phenotype involves its ability to induce the stem cell factor SOX2. IMP3 does not interact with SOX2 mRNA significantly or regulate SOX2 expression directly. We discovered that IMP3 binds avidly to SNAI2 ( SLUG ) mRNA and regulates its expression by binding to the 5' UTR. This finding is significant because SLUG has been implicated in breast CSCs and TNBC. Moreover, we show that SOX2 is a transcriptional target of SLUG. These data establish a novel mechanism of breast tumor initiation involving IMP3 and they provide a rationale for its association with aggressive disease and poor outcome.

Endoplasmic reticulum (ER) stress-mediated cell death has an important role in the pathogenesis of chronic diseases, including diabetes and neurodegeneration. Although proapoptotic programs activated by ER stress have been extensively studied, identification and characterization of antiapoptotic programs that counteract ER stress are currently incomplete. Through the gene expression profiling of β -cells lacking Wolfram syndrome 1 gene (WFS1), a causative gene for Wolfram syndrome, we discovered a novel antiapoptotic gene of the unfolded protein response (UPR), apoptosis antagonizing transcription factor (AATF). Here, we study the regulation of AATF, identify its target genes, and determine the basis for its antiapoptotic activities in response to ER stress. We show that AATF is induced by ER stress through the PERK–eIF2 α pathway and transcriptionally activates the v-akt murine thymoma viral oncogene homolog 1 (AKT1) gene through signal transducer and activator of transcription 3 (Stat3), which sustains Akt1 activation and promotes cell survival. Ectopic expression of AATF or a constitutively active form of AKT1 confers on cells resistance to ER stress-mediated cell death, whereas RNAi-mediated knockdown of AATF or AKT1 renders cells sensitive to ER stress. We also discovered a positive crosstalk between the AATF and WFS1 signaling pathways. Thus, WFS1 deficiency or AATF deficiency mediates a self-perpetuating cycle of cell death. Our results reveal a novel antiapoptotic program relevant to the treatment of diseases caused by ER stress-mediated cell death.

Combined treatment with allogeneic small lymphocytes or T-depleted small lymphocytes plus a blocking antibody to CD40 ligand (CD40L) permitted indefinite pancreatic islet allograft survival in 37 of 40 recipients that differed from islet donors at major and minor histocompatibility loci. The effect of the allogeneic small lymphocytes was donor antigen-specific. Neither treatment alone was as effective as combined treatment, although anti-CD40L by itself allowed indefinite islet allograft survival in 40% of recipients. Our interpretation is that small lymphocytes expressing donor antigens in the absence of appropriate costimulatory signals are tolerogenic for alloreactive host cells. Anti-CD40L antibody may prevent host T cells from inducing costimulatory signals in donor lymphocytes or islet grafts.

NOD mice have a generalized defect in their response to transplantation tolerance induction. T G Markees , D V Serreze , N E Phillips , C H Sorli , E J Gordon , L D Shultz , R J Noelle , B A Woda , D L Greiner , J P Mordes and A A Rossini Diabetes Division, University of Massachusetts Medical School, Worcester 01605, USA. Abstract A protocol consisting of a single donor-specific transfusion (DST) plus a brief course of anti-CD154 monoclonal antibody (anti-CD40 ligand mAb) induces permanent islet allograft survival in chemically diabetic mice, but its efficacy in mice with autoimmune diabetes is unknown. Confirming a previous report, we first observed that treatment of young female NOD mice with anti-CD154 mAb reduced the frequency of diabetes through 1 year of age to 43%, compared with 73% in untreated controls. We also confirmed that spontaneously diabetic NOD mice transplanted with syngeneic (NOD-Prkdc(scid)/Prkdc(scid)) or allogeneic (BALB/c) islets rapidly reject their grafts. Graft survival was not prolonged, however, by pretreatment with either anti-CD154 mAb alone or anti-CD154 mAb plus DST. In addition, allograft rejection in NOD mice was not restricted to islet grafts. Anti-CD154 mAb plus DST treatment failed to prolong skin allograft survival in nondiabetic male NOD mice. The inability to induce transplantation tolerance in NOD (H2g7) mice was associated with non-major histocompatibility complex (MHC) genes. Treatment with DST and anti-CD154 mAb prolonged skin allograft survival in both C57BL/6 (H2b) and C57BL/6.NOD-H2g7 mice, but it was ineffective in NOD, NOD.SWR-H2q, and NOR (H2g7) mice. Mitogen-stimulated interleukin-1beta production by antigen-presenting cells was greater in strains susceptible to tolerance induction than in the strains resistant to tolerance induction. The results suggest the existence of a general defect in tolerance mechanisms in NOD mice. This genetic defect involves defective antigen-presenting cell maturation, leads to spontaneous autoimmune diabetes in the presence of the H2g7 MHC, and precludes the induction of transplantation tolerance irrespective of MHC haplotype. Promising islet transplantation methods based on overcoming the alloimmune response by interference with costimulation may require modification or amplification for use in the setting of autoimmune diabetes.

Inbred C.B-17-scid/scid mice accept human peripheral blood mononuclear cell (PBMC) xenografts and are susceptible to human immunodeficiency virus type 1 (HIV-1) infection, but low levels of PBMC engraftment impede use of this system in HIV research. This report describes the effect of host strain background on human PBMC engraftment and HIV infectivity in scid mice. Backcrossing the scid mutation to the NOO/Lt strain (designated NOO/LtSz-scid/scid) increased the percentage of engrafted human PBMC in recipient spleens by 5- to l0-fold compared with that in C.B-17-scid/scid stock. Four weeks after human PBMC-injected mice were infected with HIV-1, 79% of NOO/LtSz-scid/scid spleens harbored replicating virus compared with only 39% of spleens in C.B-17-scid/scid mice. The NOO/LtSz-scid/scid mouse may provide a useful small animal model for studies of HIV-1.

Aims/hypothesis To develop and validate a new immunodeficient mouse strain that spontaneously develops a non-autoimmune hyperglycaemia to serve as a diabetic host for human islets and human beta stem and progenitor cells without the need for induction of hyperglycaemia by toxic chemicals with their associated side effects. Methods We generated and characterised a new strain of immunodeficient spontaneously hyperglycaemic mice, the NOD-Rag1 null Prf1 null Ins2 Akita strain and compared this strain with the NOD-scid Il2rγ null (also known as Il2rg) immunodeficient strain rendered hyperglycaemic by administration of a single dose of streptozotocin. Hyperglycaemic mice were transplanted with human islets ranging from 1,000 to 4,000 islet equivalents (IEQ) and were monitored for normalisation of blood glucose levels. Results NOD-Rag1 null Prf1 null Ins2 Akita mice developed spontaneous hyperglycaemia, similar to Ins2 Akita -harbouring strains of immunocompetent mice. Histological examination of islets in the host pancreas validated the spontaneous loss of beta cell mass in the absence of mononuclear cell infiltration. Human islets transplanted into spontaneously diabetic NOD-Rag1 null Prf1 null Ins2 Akita and chemically diabetic NOD-scid Il2rγ null mice resulted in a return to euglycaemia that occurred with transplantation of similar beta cell masses. Conclusions/interpretation The NOD-Rag1 null Prf1 null Ins2 Akita mouse is the first immunodeficient, spontaneously hyperglycaemic mouse strain described that is based on the Ins2 Akita mutation. This strain is suitable as hosts for human islet and human beta stem and progenitor cell transplantation in the absence of the need for pharmacological induction of diabetes. This strain of mice also has low levels of innate immunity and can be engrafted with a human immune system for the study of human islet allograft rejection.

Aims/hypothesis To develop and validate a new immunodeficient mouse strain that spontaneously develops a non-autoimmune hyperglycaemia to serve as a diabetic host for human islets and human beta stem and progenitor cells without the need for induction of hyperglycaemia by toxic chemicals with their associated side effects. Methods We generated and characterised a new strain of immunodeficient spontaneously hyperglycaemic mice, the NOD- Rag1 null Prf1 null Ins2 Akita strain and compared this strain with the NOD- scid Il2rγ null (also known as Il2rg ) immunodeficient strain rendered hyperglycaemic by administration of a single dose of streptozotocin. Hyperglycaemic mice were transplanted with human islets ranging from 1,000 to 4,000 islet equivalents (IEQ) and were monitored for normalisation of blood glucose levels. Results NOD- Rag1 null Prf1 null Ins2 Akita mice developed spontaneous hyperglycaemia, similar to Ins2 Akita -harbouring strains of immunocompetent mice. Histological examination of islets in the host pancreas validated the spontaneous loss of beta cell mass in the absence of mononuclear cell infiltration. Human islets transplanted into spontaneously diabetic NOD- Rag1 null Prf1 null Ins2 Akita and chemically diabetic NOD- scid Il2rγ null mice resulted in a return to euglycaemia that occurred with transplantation of similar beta cell masses. Conclusions/interpretation The NOD- Rag1 null Prf1 null Ins2 Akita mouse is the first immunodeficient, spontaneously hyperglycaemic mouse strain described that is based on the Ins2 Akita mutation. This strain is suitable as hosts for human islet and human beta stem and progenitor cell transplantation in the absence of the need for pharmacological induction of diabetes. This strain of mice also has low levels of innate immunity and can be engrafted with a human immune system for the study of human islet allograft rejection.

Key Points Severely immunodeficient mice engrafted with functional human cells and tissues, known as 'humanized' mice, facilitate progress in studies of human haematopoiesis, immunity, gene therapy, infectious diseases, cancer and regenerative medicine. Mice homozygous for the severe combined immunodeficiency ( scid ) gene mutation or for targeted mutations at the recombination-activating gene 1 ( Rag1 ) or Rag2 loci, that also have a targeted mutation at the interleukin-2 receptor γ-chain ( Il2rg ) locus, support high levels of engraftment and function of human haematopoietic stem cells (HSCs) and human immune systems. Advances in humanized mice over the past few years have included approaches to decrease host innate immune responses. In addition, humanized mouse models have benefited greatly from the identification of human species-specific molecules that are crucial for the engraftment and function of human haematopoietic and immune systems and the expression of these molecules in the immunodeficient recipient. The development of humanized mice with functional human immune systems (generated by the engraftment of human lymphoid tissues, HSCs or peripheral blood mononuclear cells) provides an opportunity to carry out translational research on human immunity and autoimmune diseases, and for the study of the biology of the human pathogens responsible for AIDS and several other human-specific infectious diseases. Humanized mice are being used as hosts for primary human tumours for studies of tumour growth and metastasis and for experimental cancer therapy. The phenotypical and functional characterization of human tumour stem cells is also being advanced through the study of humanized mice. The potential for new advances in our understanding of human immunology and other areas of human biology that are supported by studies in humanized mice remains promising. Additional genetic and technological modifications continue to accelerate progress towards the development of a robust functional human immune system in humanized mice. This article provides a comprehensive overview of the recent advances in the development and use of humanized mice. The authors consider the remaining challenges and the potential for new advances in our understanding of human immunology through the use of these mice. Significant advances in our understanding of the in vivo functions of human cells and tissues and the human immune system have resulted from the development of 'humanized' mouse strains that are based on severely immunodeficient mice with mutations in the interleukin-2 receptor common γ-chain locus. These mouse strains support the engraftment of a functional human immune system and permit detailed analyses of human immune biology, development and functions. In this Review, we discuss recent advances in the development and utilization of humanized mice, the lessons learnt, the remaining challenges and the promise of using humanized mice for the in vivo study of human immunology.